This project explores the structure and biochemistry of the phagocyte NADPH oxidase. A basic understanding of the biochemistry of this electron transport chain is critical to development of novel agents to modulate the inflammatory response. The focus is on the structure of the phagocyte-specific cytochrome b558 which we showed is a complex flavocytochrome comprised of two subunit proteins, gp91phox and p22phox. Despite knowledge of the primary structure of the cytochrome subunits, little is understood about their biosynthesis and assembly. To begin to characterize these events we have used metabolic inhibitors of heme biosynthesis, heme deprivation, and protein and immunochemical approaches to explore regulation of cytochrome biosynthesis and processing in native and recombinant systems. Findings lay the experimental foundation for a new paradigm currently under investigation in which heme transporters or chaperone-like molecules involved in heme insertion play a regulatory role in biosynthesis of heme-containing enzymes. Studies in progress address the stoichiometry of gp91phox and p22phox subunits in the cytochrome. Both physicochemical crosslinking and recombinant engineering suggest that the cytochrome is a heterotrimer comprised of two heme-bearing p22phox subunits, and a single flavin- bearing gp91phox. Findings challenge the previously accepted view of the cytochrome as a simple gp91phox-p22phox heterodimer and provide fresh insights into the search for the cytochrome heme binding domains. Oligopeptides which mimic probable contact sites between gp91phox and p47phox are being developed as candidate inhibitors of oxidase assembly.